PROJECT SUMMARY G protein-coupled receptor (GPCR) signaling plays a critical role in many physiological processes and is also involved in many human diseases, yet the mechanisms governing GPCR signaling remain poorly understood. The goal of the proposed research is to fill in gaps in knowledge regarding these mechanisms because we believe that elucidating novel aspects of GPCR signaling could identify new targets for drug development. The focus here will be on the therapeutically relevant GPCR called chemokine receptor C-X-C-receptor 4 (CXCR4). CXCR4 signaling is important for embryogenesis, immune function and stem cell regulation, among others. In addition, CXCR4 signaling is involved in several human diseases, including cancer. CXCR4 is aberrantly expressed in many cancers and its expression correlates with poor prognosis. This is mainly because CXCR4 signaling contributes to metastatic disease, the reason for most cancer related deaths. Yet the mechanisms governing CXCR4 signaling remain poorly understood. The present proposal seeks to fill in these knowledge gaps. Metastasis mediated by CXCL12/CXCR4 occurs via migration and/or survival of tumor cells. Several signaling pathways have been implicated in these processes, including the Akt signaling pathway. Akt is a serine/threonine kinase belonging to the AGC family of kinases. It is fully activated by PDK1 (PtdIns(3,4,5)P3- dependent protein kinase 1) phosphorylation at Thr-308, which is located in the kinase domain, and mTORC2 (mammalian or mechanistic target of rapamycin complex 2) phosphorylation at Ser-473, which is located in the C-terminal hydrophobic motif. One mTORC2 subunit, called DEPTOR (DEP-domain containing mTOR- interacting protein), is an inhibitor of its kinase activity. We recently showed that CXCR4 signaling promotes DEPTOR dissociation from mTORC2 and its subsequent lysosomal degradation, which is linked to mTORC2 activation and phosphorylation of Akt at Ser-473 for subsequent signaling. Yet the spatial and temporal regulation of DEPTOR by CXCR4 signaling leading to mTORC2 activation and the functional relevance of Akt signaling remain unknown. The proposed studies seek to fill in this knowledge gap. To address this we will test the hypothesis that ubiquitin-dependent DEPTOR degradation in lysosomes regulates GPCR promoted mTORC2 activity and Akt survival signaling from the surface of endosomes. To test this hypothesis three specific aims are proposed: Aim 1. To elucidate the role of AMSH in Akt signaling by CXCR4; Aim 2. To identify the mechanism by which mTORC2 is recruited to and activated at the surface of endosomes by CXCR4; and Aim 3. To elucidate the functional role of Akt signaling from the surface of endosomes. Elucidating the mechanisms by which CXCR4 promotes Akt-dependent survival signaling is highly significant because it will provide a novel conceptual understanding of CXCR4 and GPCR promoted Akt signaling, as well as provide the exciting potential to translate this knowledge into innovative drug discovery efforts to selectively target Akt signaling downstream of CXCR4 in cancer.